A fundamental problem in neurobiology concerns the generation and maintenance of neuronal identity as determined by morphology, connectivity, adhesive properties, neurotransmitter phenotype, and membrane excitability. These properties are established and maintained through the action of transcription factors. Despite significant advances in the characterization of transcription factors and of genes encoding effector molecules, the relationship between transcription factors and the expression of effector molecules that direct neuronal morphogenesis or later aspects of the neuronal phenotype such as specialized synaptic signaling properties and neurotransmitter phenotype, remains largely undefined. Here, we propose to study the role of the Drosophila Pax-6 homolog eyeless in postmitotic mushroom body neuron differentiation to provide better insights into the mechanisms by which transcription factors drive expression of neuronal traits and determine neuronal specificity. To this end, we will detail the phenotypes of postmitotic mushroom body neurons, and study the gene regulatory network controlled by eyeless. Specifically we will pursue the following specific aims. (1) Determine when eyeless is required during mushroom body development, and analyze the mushroom body defects observed in eyeless mutants. (2) Identify cis-regulatory elements required for mushroom body-specific gene expression. (3) Initiate the characterization of a putative target gene. The proposed research is essential for understanding how the cellular properties of mushroom body neurons are established by the gene regulatory network controlled by eyeless, as a prerequisite to understanding mushroom body-mediated behavior namely learning and memory and locomotion control. Furthermore, the remarkable conservation of the Pax-6 genes implies that analyzing Pax-6/eyeless function in the Drosophila brain will help explain the congenital brain defects observed in human PAX6 mutants. In addition, fundamental insights into neuronal gene regulatory networks will be important to understanding the many brain-related birth defects in children.